- Email: [email protected]
Effect of low concentration of endosulfan in vivo on cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus splenocytes
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
processes, such as immune system process, metabolic process, energy production, organelle and cytoskeleton organization, cell cycle, signal transduction, death, cell adhesion, response to stimulus and so on, suggesting that WSSV propagation after temperature rising had a comprehensive influences on the host. Almost all the DEGs related to energy production were up-regulated in Group WT, suggesting an increased energy need and a boosted energy production. Almost all the DEGs related to cell cycle were down-regulated in Group WT, implying that the host cell proliferation was inhibited. The DEGs related to positive regulation of cell death were down-regulated in Group WT, implying that WSSV inhibited the host cell death to benefit its own propagation. Through analyzing the DEGs in glycometabolism pathways, a glycometabolism pathway transition from TCA to PPP was suggested. Most host genes involved in DNA replication and repair, except one DNA ligase, were down-regulated in Group WT, suggesting an inhibited state of replication and repair of host DNA and the up-regulated DNA ligase may be involved in WSSV replication. Some DEGs were further confirmed by real-time PCR in the three groups, providing important evidence to understand the mechanisms of WSS outbreak resulted from temperature variation. * Corresponding authors. E-mail addresses: [email protected] (Y. Sun), [email protected] (J. Xiang)
P-316. Phenotypic and genotypic characterization of Streptococcus agalactiae isolates from cultured tilapia in Thailand A. Suwannasang 1, 2, *,x, N. Suanyuk 1, x, C. Tantikitti 3. 1 Kidchakan Supamattaya Aquatic Animal Health Research Center, Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla, Thailand; 2 Aquaculture Program, Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Thailand; 3 Center of Excellence in Agricultural and Natural Resources Biotechnology, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla, Thailand
Abstract Streptococcus agalactiae (Group B Streptococcus; GBS) is a leading cause of fish meningoencephalitis. The aim of this study was to characterize GBS isolated from diseased tilapia (n ¼ 13) cultured in Thailand. The bacteria was identified as GBS by conventional and rapid identification systems, multiplex PCR, and 16S rRNA analysis. The 16S rRNA gene sequence alignment showed 99% identity to the 16S rRNA sequence of several GBS strains in GenBank. Serotyping indicated that the bacteria was serotype Ia (9 isolates) and serotype III (4 isolates). Determination of virulence genes associated with GBS virulence including lmb (laminin-binding protein), scpB (C5a-peptidase), bac (beta C protein), bca (alpha C protein) and GBSi1 (Group II intron) demonstrated that genetic profiles were different within and between distinct serotype. These genotypic characteristics could be used for future study on the development of appropriate vaccine against GBS infection. * Corresponding author. E-mail address: [email protected] (A. Suwannasang) xThese authors have contributed equally to this work.
P-447. Effects of cortisol on innate immune responses and on mRNA levels of corticosteroid receptors in rainbow trout M. Teles 1, 2, *, R. Tridico 2, R. Cortes 2,3, L. Acerete 2, A. Callol 1, 4, C. Fierro-Castro 2, L. Tort 2. 1 Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; 2 Department of Cell Biology, Physiology and Immunology, UAB, 08193 Barcelona, Spain;
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3 Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC), Castellón, Spain; 4 Department of Microbiology and Ecology, Faculty of Biology, University of Valencia, 46100 Valencia, Spain
Abstract Cortisol is a key hormone in the fish stress response with a well known ability to regulate several physiological functions, including carbohydrate metabolism and the immune system. The consequences of cortisol actions in tissues are performed through the corticosteroid receptors (CRs) that have been characterized in fish during the last years. However, data concerning cortisol effects on fish innate immune system and on the transcriptional pattern of the CRs using a more controlled increase in cortisol levels isolated from any other stress related signaling is scarce. Keeping in mind the previous findings, in the present study we injected rainbow trout with slowrelease cortisol implants to create medium to high levels of circulating cortisol over extended time periods emulating an acute to chronic stress. Ten days after implantation the cortisol levels in plasma returned to control levels, emulating the period of fish recovery after stress. Glucose and lactate were also returned to control levels corroborating the recovery of the fish after cortisol injection. Thus, the present research work describes the effect of cortisol on selected innate immune responses, such as lysozyme and complement (ACH50) activities, during the recovery period. The mRNA levels of lysozyme and complement C3, factor H and factor B were measured in liver. Furthermore, we present the results concerning the transcriptional levels of the CRs in several organs of trout after 10 days implantation. The results revealed that when plasma cortisol returned to basal levels, lysozyme and ACH50 activities were significantly decreased. These results reflect a latter down-regulatory effect of cortisol on the activity of both ACH50 and lysozyme, which is in agreement with previous findings in stressed fish. In the liver, the mRNA levels of lysozyme, factor B and factor H were also significantly decreased in the recovery period. These changes in mRNA abundances correlate with the plasma data, since both lysozyme and complement activities decreased. Thus, these results suggest that cortisol regulates the expression of lysozyme and complement factors in the liver and that this effect would possibly lead to reduced plasma lysozyme and ACH50 activities. Overall, results support the relevant role of cortisol in modulating key components of the innate immune response in fish, such lysozyme or ACH50 activities, and the expression of their related genes. We also show that even in the recovery period we still found changes in the transcriptional levels of the CRs in gills, spleen and gonads. * Corresponding author. E-mail address: [email protected] (M. Teles)
P-372. Effect of low concentration of endosulfan in vivo on cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus splenocytes M.C. Tellez-Bañuelos 1, P.C. Ortiz 2, L.F. Jave 2, V.H. Siordia 1, 2, A. Santerre 1, *, G.P. Zaitseva 1. 1 Departamento de Biología Celular y Molecular, Universidad de Guadalajara, Carretera a Nogales Km 15.5, z.p. 45110, Las Agujas, Zapopan, Jalisco, Mexico; 2 Centro de Investigación Biomédica de Occidente, IMSS, Sierra Mojada 800, Col. Independencia, z.p. 44340, Guadalajara, Jalisco, Mexico
Abstract Endosulfan is a potent organochlorinated pesticide restricted in many countries for its adverse effects on ecosystems, including aquatic environments. Data from our research group indicated that endosulfan induces oxidative stress and non-specific activation of splenic macrophages, and exacerbates serum interleukin-2 synthesis in Oreochromis niloticus (Nile tilapia). Endosulfan per se increases cell proliferation, but decreases the lymphoproliferative response to mitogenic stimulus. Splenocytes exposed in vitro to endosulfan for 15–180 min show significantly higher levels of pERK1/2 than non-exposed control cells. Moreover, endosulfan mediates a decrease in etoposide-induced apoptosis and provokes cell senescence. In
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Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
teleosts, nonspecific cytotoxic cells (NCC) have the ability to kill a wide array of targets including virus-infected cells, cancer cells (HL-60), and parasites. Granzymes are pore-forming proteins which constitute the major components of granules of professional killer cells such as cytotoxic T lymphocytes, natural killer cells and NCC. Here, the effect of the acute in vivo exposure of juvenile Nile tilapia to ½ LC50 of endosulfan (7 ppb for 96 h) on the percentage and activity of splenic NCC against HL-60 tumor cells, and the expression of granzyme, were investigated by using flow cytometry and real-time polymerase chain reaction (RT-PCR). NCC were purified from spleen of exposed and control fish, by gradient centrifugation over Ficoll histopaque followed by isolation over Percoll. Immunophenotype and percentage of purified NCC were confirmed through the specific union of the monoclonal antibody 5C6 and FITC fluorescence detection Cytotoxic activity was assessed by setting up 4 hours co-cultures of NCC and HL-60 tumoral cells, using the following proportions of target (T) and effector (E) cells: 1:2, 1:10, 1:20 and 1:40. For RT-PCR, specific primers were designed from cDNA sequences of granzyme and b-actin genes of O. niloticus. The exposure of Nile tilapia to endosulfan showed a tendency in the increase of the percentage of FITC+ NCC (4,23%) and a significant decrease in cytotoxic activity (73,11%) when using a 1:40 T:E proportion. The expression of the granzime gene was significantly lower in cells from exposed fish compared to non-exposed ones (69,5% decrease). The present study demonstrates that acute in vivo exposure to endosulfan deregulates NCC splenic function which may facilitate the development of virus relateddiseases. Thus it is important to create conscience on the immunological risks of organochlorinated pesticides in immune innate cellular response in fish and other organisms. * Corresponding author. E-mail address: [email protected] (A. Santerre)
P-238. First evidence of a Toll signaling pathway involved in innate immune response in Lophotrochozoa M. Toubiana 1, *, M. Gerdol 2, U. Rosani 3, A. Pallavicini 2, P. Venier 3, P. Roch 1. 1
Ecologie des Systèmes Marins et Côtiers (EcoSym), Université Montpellier 2-CNRS, cc 093, Place E. Bataillon, F-34095 Montpellier cedex 05, France; 2 University of Trieste, Department of Life Science, Laboratory of Genetics, Via Licio Giorgieri 5, 34127 Trieste (TS), Italy; 3 Department of Biology, University of Padua, Via U. Bassi, 58/B, 35121 Padova, Italy Abstract Antimicrobial peptides (AMPs) are certainly one of the major effectors of the anti-infectious innate immunity. The complete molecular cascade of signal transduction from pathogen recognition to AMP activity is only known in Drosophila (Ecdysozoa), although not totally elucidated. Only some of the molecular intermediates have been reported from Lophotrochozoa, mainly as partial cDNA sequences. In mussels, biochemical analysis completed by molecular approaches, reveal a huge diversity of AMPs, including within the same animal, suggesting a complex system of molecular effectors. Expression regulations and specificities have been investigated following injections with bacteria, fungi, and physical stresses. No strict correlation has been yet established between structure and function of AMPs in mussels. Recent scientific advances in term of high-speed sequencing have allowed to obtain a large mussel EST database, completed with Illumina reads assemblies. The scanning of these libraries, based on protein's conserved domains (by analogy with vertebrates and Drosophila) confirmed the presence of molecules involved in recognition-signal transduction and revealed new ones. First discovery was the existence in the mussel, Mytilus galloprovincialis, of 23 Toll-like receptors (TLRs) and 3 myeloid differentiation factors 88 (MyD88s).
These TLRs possess the characteristic canonical structure with the presence of extracellular leucine-rich repeat (LRR) domains in variable number, followed by a transmembrane region and an intracellular part composed by a Toll-interleukine 1 receptor (TIR) domain. Study of LRR structure revealed that only 3 TLR present multiple cysteine clusters (mccTLR), found in the Toll receptor of Ecdysozoa (Drosophile Toll-1). The similarities observed in up regulation of gene expression of only one mccTLR and one MyD88 in response to microorganism injection gave some light on the existence of the cascade in mussel. We recently identified most of the other members of the NF-kB cascade. Their structures and gene expressions will be presented and discussed. In conclusion, Lophotrochozoa, as well as Ecdysozoa, appeared to rely on the NF-kB pathway for signal transduction. However, the functionality of such complex system in the course of real infection is still to be fully understood. This work was partially funded by the EU program BIVALIFE (KBBE-2010266157). * Corresponding author. E-mail address: [email protected] (M. Toubiana)
P-268. Adjuvant capacity of the aromatic geranyl derivative Filifolinone in salmonids B. Valenzuela 1, *, F.E. Rodríguez 1, J. Obreque 1, B. Modak 2, M. Imarai 1, *. 1 Department of Biology, Center for Research in Aquatic Biotechnology, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile; 2 Department of Natural Products, Center for Research in Aquatic Biotechnology, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile
Abstract 3 H-Spiro [1-benzofuran-2,10 -ciclohexane] called Filifolinone is a semisynthetic derivative obtained from Filifolinol extracted of Helitropium filifolium. Interestingly, Filifolinone increases the levels of expression of pro-inflammatory and anti-inflammatory cytokines in the cell line SHK-1 and in salmonid fish. Because of these immunostimulatory properties, we evaluated the adjuvant activity in the rainbow trout. Fish were immunized twice with ovalbumin (OVA) and Filifolinone. After 20 days, spleens were removed and used to evaluate the OVA-dependent proliferation by tritiated thymidine incorporation and by CD4+ cell quantitation by flow cytometry. Cytokine expression was also analyzed by real-time reversetranscription PCR (RT-PCR). Kidneys of each fish were also obtained and used to analyze the levels of transcripts of interferon gamma (IFNg), interleukin (IL)4/13 and IL-17. In addition, transcripts of transcription factors T-bet, STAT1, GATA3 and RORgT and T cell co-receptors CD4 and CD8 alpha were also examined. Results showed that Filifolinone enhanced the OVA-induced splenocyte proliferation in the immunized fish (P < 0.05). Proliferating cells were CD4+ lymphoid cells and showed enhanced levels of IFNg, IL4/13, MHC class II and CD4 in regard to the control splenocytes. In addition, mRNA levels of IFNg, T-bet, GATA3 and STAT1 and the cell surface marker, CD4 also increased in the kidneys of fish immunized with OVA and Filifolinone compared to control fish immunized with OVA alone. Overall, results demonstrated that Filifolinone enhanced significantly the cellular response against OVA and seems to induce a Th1 as well as Th2 type immune response in the rainbow trout. Acknowledge: INNOVA-CHILE 07CN13PBT-90, INNOVA-CHILE 09MCSS6698, FONDECYT 1110295, Becas CONICYT. * Corresponding authors. E-mail addresses: [email protected] (B. Valenzuela), monica.imarai@usach. cl (M. Imarai)
processes, such as immune system process, metabolic process, energy production, organelle and cytoskeleton organization, cell cycle, signal transduction, death, cell adhesion, response to stimulus and so on, suggesting that WSSV propagation after temperature rising had a comprehensive influences on the host. Almost all the DEGs related to energy production were up-regulated in Group WT, suggesting an increased energy need and a boosted energy production. Almost all the DEGs related to cell cycle were down-regulated in Group WT, implying that the host cell proliferation was inhibited. The DEGs related to positive regulation of cell death were down-regulated in Group WT, implying that WSSV inhibited the host cell death to benefit its own propagation. Through analyzing the DEGs in glycometabolism pathways, a glycometabolism pathway transition from TCA to PPP was suggested. Most host genes involved in DNA replication and repair, except one DNA ligase, were down-regulated in Group WT, suggesting an inhibited state of replication and repair of host DNA and the up-regulated DNA ligase may be involved in WSSV replication. Some DEGs were further confirmed by real-time PCR in the three groups, providing important evidence to understand the mechanisms of WSS outbreak resulted from temperature variation. * Corresponding authors. E-mail addresses: [email protected] (Y. Sun), [email protected] (J. Xiang)
P-316. Phenotypic and genotypic characterization of Streptococcus agalactiae isolates from cultured tilapia in Thailand A. Suwannasang 1, 2, *,x, N. Suanyuk 1, x, C. Tantikitti 3. 1 Kidchakan Supamattaya Aquatic Animal Health Research Center, Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla, Thailand; 2 Aquaculture Program, Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Thailand; 3 Center of Excellence in Agricultural and Natural Resources Biotechnology, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla, Thailand
Abstract Streptococcus agalactiae (Group B Streptococcus; GBS) is a leading cause of fish meningoencephalitis. The aim of this study was to characterize GBS isolated from diseased tilapia (n ¼ 13) cultured in Thailand. The bacteria was identified as GBS by conventional and rapid identification systems, multiplex PCR, and 16S rRNA analysis. The 16S rRNA gene sequence alignment showed 99% identity to the 16S rRNA sequence of several GBS strains in GenBank. Serotyping indicated that the bacteria was serotype Ia (9 isolates) and serotype III (4 isolates). Determination of virulence genes associated with GBS virulence including lmb (laminin-binding protein), scpB (C5a-peptidase), bac (beta C protein), bca (alpha C protein) and GBSi1 (Group II intron) demonstrated that genetic profiles were different within and between distinct serotype. These genotypic characteristics could be used for future study on the development of appropriate vaccine against GBS infection. * Corresponding author. E-mail address: [email protected] (A. Suwannasang) xThese authors have contributed equally to this work.
P-447. Effects of cortisol on innate immune responses and on mRNA levels of corticosteroid receptors in rainbow trout M. Teles 1, 2, *, R. Tridico 2, R. Cortes 2,3, L. Acerete 2, A. Callol 1, 4, C. Fierro-Castro 2, L. Tort 2. 1 Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; 2 Department of Cell Biology, Physiology and Immunology, UAB, 08193 Barcelona, Spain;
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3 Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC), Castellón, Spain; 4 Department of Microbiology and Ecology, Faculty of Biology, University of Valencia, 46100 Valencia, Spain
Abstract Cortisol is a key hormone in the fish stress response with a well known ability to regulate several physiological functions, including carbohydrate metabolism and the immune system. The consequences of cortisol actions in tissues are performed through the corticosteroid receptors (CRs) that have been characterized in fish during the last years. However, data concerning cortisol effects on fish innate immune system and on the transcriptional pattern of the CRs using a more controlled increase in cortisol levels isolated from any other stress related signaling is scarce. Keeping in mind the previous findings, in the present study we injected rainbow trout with slowrelease cortisol implants to create medium to high levels of circulating cortisol over extended time periods emulating an acute to chronic stress. Ten days after implantation the cortisol levels in plasma returned to control levels, emulating the period of fish recovery after stress. Glucose and lactate were also returned to control levels corroborating the recovery of the fish after cortisol injection. Thus, the present research work describes the effect of cortisol on selected innate immune responses, such as lysozyme and complement (ACH50) activities, during the recovery period. The mRNA levels of lysozyme and complement C3, factor H and factor B were measured in liver. Furthermore, we present the results concerning the transcriptional levels of the CRs in several organs of trout after 10 days implantation. The results revealed that when plasma cortisol returned to basal levels, lysozyme and ACH50 activities were significantly decreased. These results reflect a latter down-regulatory effect of cortisol on the activity of both ACH50 and lysozyme, which is in agreement with previous findings in stressed fish. In the liver, the mRNA levels of lysozyme, factor B and factor H were also significantly decreased in the recovery period. These changes in mRNA abundances correlate with the plasma data, since both lysozyme and complement activities decreased. Thus, these results suggest that cortisol regulates the expression of lysozyme and complement factors in the liver and that this effect would possibly lead to reduced plasma lysozyme and ACH50 activities. Overall, results support the relevant role of cortisol in modulating key components of the innate immune response in fish, such lysozyme or ACH50 activities, and the expression of their related genes. We also show that even in the recovery period we still found changes in the transcriptional levels of the CRs in gills, spleen and gonads. * Corresponding author. E-mail address: [email protected] (M. Teles)
P-372. Effect of low concentration of endosulfan in vivo on cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus splenocytes M.C. Tellez-Bañuelos 1, P.C. Ortiz 2, L.F. Jave 2, V.H. Siordia 1, 2, A. Santerre 1, *, G.P. Zaitseva 1. 1 Departamento de Biología Celular y Molecular, Universidad de Guadalajara, Carretera a Nogales Km 15.5, z.p. 45110, Las Agujas, Zapopan, Jalisco, Mexico; 2 Centro de Investigación Biomédica de Occidente, IMSS, Sierra Mojada 800, Col. Independencia, z.p. 44340, Guadalajara, Jalisco, Mexico
Abstract Endosulfan is a potent organochlorinated pesticide restricted in many countries for its adverse effects on ecosystems, including aquatic environments. Data from our research group indicated that endosulfan induces oxidative stress and non-specific activation of splenic macrophages, and exacerbates serum interleukin-2 synthesis in Oreochromis niloticus (Nile tilapia). Endosulfan per se increases cell proliferation, but decreases the lymphoproliferative response to mitogenic stimulus. Splenocytes exposed in vitro to endosulfan for 15–180 min show significantly higher levels of pERK1/2 than non-exposed control cells. Moreover, endosulfan mediates a decrease in etoposide-induced apoptosis and provokes cell senescence. In
1742
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
teleosts, nonspecific cytotoxic cells (NCC) have the ability to kill a wide array of targets including virus-infected cells, cancer cells (HL-60), and parasites. Granzymes are pore-forming proteins which constitute the major components of granules of professional killer cells such as cytotoxic T lymphocytes, natural killer cells and NCC. Here, the effect of the acute in vivo exposure of juvenile Nile tilapia to ½ LC50 of endosulfan (7 ppb for 96 h) on the percentage and activity of splenic NCC against HL-60 tumor cells, and the expression of granzyme, were investigated by using flow cytometry and real-time polymerase chain reaction (RT-PCR). NCC were purified from spleen of exposed and control fish, by gradient centrifugation over Ficoll histopaque followed by isolation over Percoll. Immunophenotype and percentage of purified NCC were confirmed through the specific union of the monoclonal antibody 5C6 and FITC fluorescence detection Cytotoxic activity was assessed by setting up 4 hours co-cultures of NCC and HL-60 tumoral cells, using the following proportions of target (T) and effector (E) cells: 1:2, 1:10, 1:20 and 1:40. For RT-PCR, specific primers were designed from cDNA sequences of granzyme and b-actin genes of O. niloticus. The exposure of Nile tilapia to endosulfan showed a tendency in the increase of the percentage of FITC+ NCC (4,23%) and a significant decrease in cytotoxic activity (73,11%) when using a 1:40 T:E proportion. The expression of the granzime gene was significantly lower in cells from exposed fish compared to non-exposed ones (69,5% decrease). The present study demonstrates that acute in vivo exposure to endosulfan deregulates NCC splenic function which may facilitate the development of virus relateddiseases. Thus it is important to create conscience on the immunological risks of organochlorinated pesticides in immune innate cellular response in fish and other organisms. * Corresponding author. E-mail address: [email protected] (A. Santerre)
P-238. First evidence of a Toll signaling pathway involved in innate immune response in Lophotrochozoa M. Toubiana 1, *, M. Gerdol 2, U. Rosani 3, A. Pallavicini 2, P. Venier 3, P. Roch 1. 1
Ecologie des Systèmes Marins et Côtiers (EcoSym), Université Montpellier 2-CNRS, cc 093, Place E. Bataillon, F-34095 Montpellier cedex 05, France; 2 University of Trieste, Department of Life Science, Laboratory of Genetics, Via Licio Giorgieri 5, 34127 Trieste (TS), Italy; 3 Department of Biology, University of Padua, Via U. Bassi, 58/B, 35121 Padova, Italy Abstract Antimicrobial peptides (AMPs) are certainly one of the major effectors of the anti-infectious innate immunity. The complete molecular cascade of signal transduction from pathogen recognition to AMP activity is only known in Drosophila (Ecdysozoa), although not totally elucidated. Only some of the molecular intermediates have been reported from Lophotrochozoa, mainly as partial cDNA sequences. In mussels, biochemical analysis completed by molecular approaches, reveal a huge diversity of AMPs, including within the same animal, suggesting a complex system of molecular effectors. Expression regulations and specificities have been investigated following injections with bacteria, fungi, and physical stresses. No strict correlation has been yet established between structure and function of AMPs in mussels. Recent scientific advances in term of high-speed sequencing have allowed to obtain a large mussel EST database, completed with Illumina reads assemblies. The scanning of these libraries, based on protein's conserved domains (by analogy with vertebrates and Drosophila) confirmed the presence of molecules involved in recognition-signal transduction and revealed new ones. First discovery was the existence in the mussel, Mytilus galloprovincialis, of 23 Toll-like receptors (TLRs) and 3 myeloid differentiation factors 88 (MyD88s).
These TLRs possess the characteristic canonical structure with the presence of extracellular leucine-rich repeat (LRR) domains in variable number, followed by a transmembrane region and an intracellular part composed by a Toll-interleukine 1 receptor (TIR) domain. Study of LRR structure revealed that only 3 TLR present multiple cysteine clusters (mccTLR), found in the Toll receptor of Ecdysozoa (Drosophile Toll-1). The similarities observed in up regulation of gene expression of only one mccTLR and one MyD88 in response to microorganism injection gave some light on the existence of the cascade in mussel. We recently identified most of the other members of the NF-kB cascade. Their structures and gene expressions will be presented and discussed. In conclusion, Lophotrochozoa, as well as Ecdysozoa, appeared to rely on the NF-kB pathway for signal transduction. However, the functionality of such complex system in the course of real infection is still to be fully understood. This work was partially funded by the EU program BIVALIFE (KBBE-2010266157). * Corresponding author. E-mail address: [email protected] (M. Toubiana)
P-268. Adjuvant capacity of the aromatic geranyl derivative Filifolinone in salmonids B. Valenzuela 1, *, F.E. Rodríguez 1, J. Obreque 1, B. Modak 2, M. Imarai 1, *. 1 Department of Biology, Center for Research in Aquatic Biotechnology, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile; 2 Department of Natural Products, Center for Research in Aquatic Biotechnology, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile
Abstract 3 H-Spiro [1-benzofuran-2,10 -ciclohexane] called Filifolinone is a semisynthetic derivative obtained from Filifolinol extracted of Helitropium filifolium. Interestingly, Filifolinone increases the levels of expression of pro-inflammatory and anti-inflammatory cytokines in the cell line SHK-1 and in salmonid fish. Because of these immunostimulatory properties, we evaluated the adjuvant activity in the rainbow trout. Fish were immunized twice with ovalbumin (OVA) and Filifolinone. After 20 days, spleens were removed and used to evaluate the OVA-dependent proliferation by tritiated thymidine incorporation and by CD4+ cell quantitation by flow cytometry. Cytokine expression was also analyzed by real-time reversetranscription PCR (RT-PCR). Kidneys of each fish were also obtained and used to analyze the levels of transcripts of interferon gamma (IFNg), interleukin (IL)4/13 and IL-17. In addition, transcripts of transcription factors T-bet, STAT1, GATA3 and RORgT and T cell co-receptors CD4 and CD8 alpha were also examined. Results showed that Filifolinone enhanced the OVA-induced splenocyte proliferation in the immunized fish (P < 0.05). Proliferating cells were CD4+ lymphoid cells and showed enhanced levels of IFNg, IL4/13, MHC class II and CD4 in regard to the control splenocytes. In addition, mRNA levels of IFNg, T-bet, GATA3 and STAT1 and the cell surface marker, CD4 also increased in the kidneys of fish immunized with OVA and Filifolinone compared to control fish immunized with OVA alone. Overall, results demonstrated that Filifolinone enhanced significantly the cellular response against OVA and seems to induce a Th1 as well as Th2 type immune response in the rainbow trout. Acknowledge: INNOVA-CHILE 07CN13PBT-90, INNOVA-CHILE 09MCSS6698, FONDECYT 1110295, Becas CONICYT. * Corresponding authors. E-mail addresses: [email protected] (B. Valenzuela), monica.imarai@usach. cl (M. Imarai)